Macropolycyclic rare earth complexes and application as fluorescent tracers

ABSTRACT

The invention relates to macropolycyclic rare earth complexes, namely cryptates which are useful as fluorescent tracers.

The present invention, which is the result of work carried out incollaboration with Professor J. M. LEHN and his research team at theUniversite Louis Pasteur in Strasbourg, relates to the field offluorescence and more particularly of fluorescent macropolycycliccomplexes which are especially suitable as tracers in immunologicaldeterminations.

It is known that detection methods using fluorescence are intrinsicallyvery sensitive and could make it possible to obtain lower detectionlimits than those reached by means of radioactivity measurements, inparticular through the use of laser sources (I. Wieder,Immunofluorescence and related staining techniques, 1978, Elsevier).

In practice, however, these detection limits are not reached because themeasurement is made in a matrix which does not have the propertiesrequired to achieve this optimum. In general, the measurement medium isturbid and favors diffusion, and other molecules fluorescing at the samewavelength can be present in the measurement medium.

In some cases, the improvements made to the measuring equipment are notsufficient to substantially improve this detection limit or are veryexpensive (laser, monochromators, etc.).

This state of things is even more troublesome in the field ofbiochemistry and immunology, where very small quantities of activemolecules have to be measured in biological media which can be turbid orcan contain proteins or other molecules which are themselves fluorescent(turbidity and intrinsic fluorescence of the serum).

In immunological determinations using a fluorescent tracer, when theantigen or antibody is labeled with the fluorescent molecule, the lattermust possess the following properties:

it must possess a chemical function which permits coupling with thebiological molecule without denaturing it or modifying its immunologicalproperties;

the molar absorption coefficient of the fluorescent molecule must be ashigh as possible;

the quantum yield of fluorescence must be the highest;

the Stokes shift must be as large as possible;

the emission wavelength must be greater than 500 nm if possible; and

it must be soluble in water or buffer solutions.

These conditions are specified for example in the article by E. SOINI inClin. Chem. 25, 353 (1979). Now, the molecules known to date are farfrom possessing all these properties.

The fluorescence of certain rare earth chelates has been known for manyyears through the work relating to their application in the field oflasers (A. P. B. SINHA, Spectroscop. Inorg. Chem. 2, 255 (1971)).

These complexes are formed of:

firstly a chelating molecule possessing an electron system capable ofpopulating the triplet state T₁ by inter-system crossing afterexcitation of the singlet state S₁ following the absorption of luminousenergy; and

secondly a rare earth ion which, in the case of fluorescenceapplications, possesses a strong ion fluorescence intensity (Eu, Tb) ora moderate intensity (Sm, Dy), and whose resonance level is populated bythe transfer of non-radiant energy from the triplet state of thecomplexing agent.

In order to obtain a strong fluorescence of the rare earth chelate, itis necessary for the triplet energy of the complexing molecule to begreater than that of the resonance level of the ion, and for it to havea sufficient life-time (preponderance of this type of deactivationcompared with other processes such as phosphorescence and non-radiantdeactivation, for example thermal deactivation).

In this case, after excitation in the absorption band of the chelate, afluorescence characteristic of the rare earth ion is observed.

These compounds have great advantages in the field of detection by themeasurement of fluorescence; for example, they are characterized by :

a large Stokes shift,

a high molar absorption coefficient, ε, relative to that of the rareearth,

the possibility of simultaneous detection of several chelates bychanging the rare earth,

an emission spectrum characteristic of the rare earth (line spectrum, λof maximum emission >500 nm), and

a long life-time (from a microsecond to a millisecond).

U.S. Pat. No. 4,058,732 describes a method of analytical spectroscopy byfluorescence which uses fluorescent molecules (rare earth chelates)having a relatively long life-time. In view of the fact that thediffusion due to the particles of the matrix, and the fluorescence ofthe majority of the organic molecules which make up the matrix, areshort-lived phenomena (generally lasting less than 1 μsec.), the saidpatent recommends a pulsed excitation and a functionalized rare earthchelate for labeling the biological molecules to be identified,detection taking place between each pulsation and after a sufficientlylong time for the undesired phenomena to have substantially decreased.

According to the prior art cited below, rare earth chelatestheoretically satisfy all the conditions for forming a class of idealfluorescent molecules, in particular as tracers for biological moleculesin immunological determinations, in cytology and for cell classifiers:

U S. Pat. No. 4,058,732

SOINI, Clin. Chem. 25, 353 (1979)

L. M. VALLARINO et al. in Automation of Uterine Cancer Cytology 2,Proceedings of 2nd Int. Conf., G. L. WIED, G. F. GAHR and P. H. BARTEL,Editors, Tutorials of Cytology, Chicago LL 1977.

Now, in practice, it is found that the chelates used hitherto do nothave all the following properties required by these applications, thesebeing mainly:

a high quantum yield of fluorescence and a high molar absorptioncoefficient,

a suitable triplet energy of the chelating agent,

non-inhibition by the solvent (water or other solvent) or by moleculespresent in the medium in which the measurement is made,

ease of functionalization for the purpose of coupling with molecules ofbiological interest or other molecules,

selectivity of the chelation in favor of the rare earth and at theexpense of other cations which may be present in large quantities in themeasurement medium,

solubility in water under the conditions of application of theimmunological determinations, and

a high stability, in particular at low dilution.

The β-diketone chelates described in U.S. Pat. No. 4,048,732 are onlysparingly soluble in water and biological media, if at all, and theirfluorescence is largely inhibited by water.

Other chelates have been proposed as tracers in immunologicaldeterminations; in particular, there may be mentioned the chelatesderived from EDTA, HEDTA and DTPA, imidodiacetate and the like, anddescribed for example in: Proc. Nat. Acad. Sci. U.S.A. 72, 4764 (1975),U.S. Pat. No. 4,374,120, European Patent Application No. A-0068875 andGerman Offenlegungsschrift No. 3 033 691. Their efficacy is assumed tooriginate from the high value of their stability constant, which isgenerally greater than 10¹⁰.

A criterion of this kind, based solely on thermodynamic considerations,is certainly not sufficient since it has been shown that the life-timeof a Tb(III) EDTA complex is increased by the addition of apotransferrin(Biochem. 19, 5057, 1980), which is characteristic of a protein-chelatebond.

It has recently been recommended, when using tracers of the Eu(III) EDTAtype such as those described in U.S. Pat. No. 4,374,120, to use a buffercontaining DTPA in order to remove the free europium which hasdissociated from the chelate, clearly proving the instability of suchchelates Clin. Chem. 29, 60, 1983).

This state of the prior art shows that the rare earth chelates employedhitherto cannot be used at low dilution in aqueous media containingother cations or in biological media, and in particular in immunologicaldeterminations, and that criteria such as the kinetic stability (rate ofdissociation of the complex) and the selectivity of formation of therare earth complex should be taken into account.

It may be noted moreover that, by virtue of their high formationconstant (in general >10¹⁰), the majority of the chelates of the priorart have found an application in technical γ imagery, which usesγ-emitting heavy ions of relatively short life-time, obtained in diluteaqueous solutions. The fixation of the ion must then take place rapidlyon the molecule of biological interest which carries the chelatingagent; in particular, the rate of formation of the chelate must be high:##STR1## k₁ and k-1 being the rate constants of formation anddissociation respectively; at equilibrium, we have: ##EQU1##

For the complexes used hitherto, and for the reasons referred to above.the chelates which have always been used make it possible to obtain ahigh value of k₁ [≃1.9·10²² M⁻¹ min.⁻¹ for Eu³⁺ EDTA--see J. inorg.nucl. Chem. 1971, 33, p. 127]. Consequently, for a given value of K, thedissociation rate constant is relatively large, which means that therate of ion exchange between the chelate and the solution is relativelyrapid.

Macropolycyclic rare earth complexes have now been found which possessexcellent properties of selectivity and stability, especially of kineticstability in aqueous media and biological media. These macropolycycliccomplexes are particularly suitable as fluorescent tracers forbiological substances in immunological detection or determinationtechniques using fluorescence. They are also suitable as reagents inluminescence reactions.

It has actually been found that it is possible to enhance thefluorescence of a rare earth ion in aqueous solution by complexing theion with a macropolycyclic compound possessing a donor unit with agreater triplet energy than the emission level of the rare earth ion,and by exciting the donor unit.

Whereas the excitation of a rare earth ion produces a very weakfluorescence because rare earths generally have low molar absorptioncoefficients ε, excitation of the donor unit of the macropolycycliccompound defined below makes it possible to enhance the fluorescencecharacteristic of the rare earth ion. The rare earth complexes thusformed are excellent fluorescent tracers; furthermore, these arecompounds which are stable in aqueous media and possess a very highselectivity in respect of rare earth ions.

In contrast to the chelates of the prior art, which are characterized bya high formation constant, the rare earth complexes according to theinvention have as essential characteristic a high kinetic stability (lowrate of dissociation). This characteristic is all the more importantbecause the biological solutions used in immunological detection anddetermination methods generally contain proteins which themselves arealso capable of fixing the rare earth ion. On the other hand, the rateof formation of the complexes of the invention is not critical. Theformation of the rare earth ion complex, on the one hand, and thecoupling of the resulting complex with the biological molecule, on theother, can be carried out separately. Consequently, the formation of therare earth ion complex according to the invention can be carried outunder non-biological conditions (use of organic solvents, possibility ofsupplying energy, time, etc.).

According to a first feature, the present invention therefore relates tothe use of macropolycyclic rare earth complexes as fluorescent tracers,especially in immunological detection and determination methods, thesaid complexes consisting of at least one rare earth salt complexed by amacropolycyclic compound of the general formula: ##STR2## in which Z isa trivalent or tetravalent atom such as nitrogen, carbon or phosphorus,R is nothing or represents hydrogen, the hydroxyl group, an amino groupor a hydrocarbon radical, and the divalent radicals ○A , ○B and ○Cindependently of one another are hydrocarbon chains which optionallycontain one or more heteroatoms and are optionally interrupted by aheteromacrocycle, at least one of the radicals ○A , ○B or ○C alsocontaining at least one molecular unit or essentially consisting of amolecular unit, the said molecular unit possessing a greater tripletenergy than the emission level of the complexed rare earth ion.

According to another feature, the invention also relates to a processfor enhancing the fluorescence of a rare earth ion, which consists incomplexing at least one rare earth ion with a macropolycyclic compound,such as defined above, possessing a molecular unit with a greatertriplet energy than the emission level of the rare earth ion, and inexciting the complex thus formed at the absorption wavelength of thesaid molecular unit.

The rare earth complexes defined above are new compounds except for theeuropium and terbium complexes obtained with the macropolycycliccompound of the formula below: ##STR3##

Thus, the present invention also relates, by way of new compounds, tothe rare earth complexes consisting of at least one rare earth saltcomplexed by a macropolycyclic compound of the formula I above, with theproviso that, if the rare earth salt is a europium or terbium salt, Z isnitrogen, ○A is --(CH₂)₂ --O--C₆ H₃ R--O--(CH₂)₂ -- and with R═H or NH₂,○B and ○C are not simultaneously the radical --(CH₂)₂ --O--(CH₂)₂--O--(CH₂)₂ -- in one case and the radical --(CH₂)₂ --O--(CH₂)₂ -- inthe other.

The hydrocarbon chains which form the radicals ○A , ○B and ○C cancontain 2 or more carbon atoms and can optionally be interrupted by oneor more heteroatoms chosen from the group consisting of oxygen, sulfuror nitrogen atoms.

The preferred hydrocarbon chains for the purposes of the invention arepolyether chains, in particular ethoxylated or polyethoxylated chains.

The molecular unit which constitutes an essential component of themacropolycyclic compound according to the invention is a triplet energydonor unit composed of molecules or groups of molecules possessing agreater triplet energy than the emission level of the complexed rareearth ion.

The energy transfer takes place from the triplet level of the donor unitto one of the emission levels of the complexed rare earth. For example,europium possesses the emission levels ⁵ D₀ at 17270 cm⁻¹, ⁵ D₁ at 19030cm⁻¹ and possibly ⁵ D₂, and terbium possesses the emission level ⁵ D₄ at20480 cm⁻¹.

The triplet energy donor units suitable for the purposes of theinvention must possess a triplet energy which is greater than or equalto that of the emission levels of the rare earth ion. For example, inthe case of the europium and terbium complexes according to theinvention, the triplet level of the donor unit must be greater than17270 cm⁻¹.

As the phenomenon of phosphorescence is due to the radiant deactivationof a triplet state, a preferred criterion for the donor units can be thephosphorescence emission wavelength of these units. For example, thechosen units will emit a phosphorescence at lower wavelengths (higherenergies) than those corresponding to the population of the emissionlevels of the rare earth. In the present case, the phosphorescencewavelength of the donor unit will have to be below 580 nm.

It is pointed out that the complexes according to the invention can haveone or more donor units, the said donor units constituting either all orpart of the radicals ○A , ○B and ○C .

Without implying a limitation, molecular units can be used are anytriplet sensitizers having the requisite triplet energy, for examplethose described in European Patent Application No. A-0068875 or inSpectroscop. Inorg. Chem. 2, 255, 1971, these documents being cited inthe present description by way of reference.

Particularly preferred molecular units for the purposes of the inventionare phenanthroline, anthracene, benzene, naphthalene, biphenyl andterphenyl, bipyridines, biquinolines such as the bisisoquinolines, forexample 2,2'-bipyridine, azobenzene, azopyridine, pyridine or2,2'-bisisoquinoline.

The chains below may be mentioned in particular as examples of radicals○A , ○B and ○C containing an energy donor unit: --C₂ H₄ --X₁ --C₆ H₄--X₂ --C₂ H₄ --; --C₂ H₄ --X₁ --CH₂ --C₆ H₄ --CH₂ --X₂ --C₂ H₄ --; X₁and X₂, which can be identical or different, denote oxygen, nitrogen orsulfur; ##STR4## X being oxygen or hydrogen.

The macropolycyclic rare earth complexes according to the invention canbe obtained by the conventional processes for the preparation of metalcomplexes, which consist in reacting the complexing compound with acompound donating the cation to be complexed. Processes of this type aredescribed especially in U.S. Pat. Nos. 3,888,377, 3,966,766 and4,156,683, which are cited in the present description by way ofreference.

For example, the macropolycyclic complexes can be obtained by reacting arare earth cation donor compound with the macropolycyclic compoundhaving the characteristics defined above, each compound advantageouslybeing in solution, preferably in the same solvent or in compatiblesolvents which are inert towards complex formation. In general,acetonitrile, DMSO or ethanol is used as the solvent.

The rare earth cation donor compound which can be used is any rare earthsalt, advantageously a chloride an acetylacetonate or a nitrate.

The reaction is advantageously carried out at the boiling point of thesolvent.

lf the macropolycyclic complex formed is soluble in the reactionsolvent, it is isolated from the solution by evaporation to dryness. lfthe macropolycyclic complex formed crystallizes from the reactionsolvent, it is separated off by filtration or any other appropriateconventional means. The complexes thus obtained can be purified bycrystallization.

The above reaction can also be carried out using a solution of themacropolycyclic compound and the cation donor compound in thecrystalline form. A synergistic agent for protecting againstdeactivation can also be introduced into the coordination sphere of thecation, as described in J. Chem. Phys. 40, 2790 (1964) and 41, 157(1964).

In the remainder of the present description, the macropolycycliccomplexes according to the invention are also called "cryptates" and themacropolycyclic compound by itself is also called "cryptand". Thenomenclature as defined by LEHN will be adopted below for denoting thecryptates and cryptands. For this purpose, reference may be madeespecially to the articles by J. M. LEHN in Struct. Bonding (Berlin) 16,1, 1973 and Acc. Chem. Res. 11, 49 (1978).

All the rare earth ions are suitable for the purposes of the presentinvention. However, preference will be given to those which exhibit themost intense ion fluorescence, i.e. terbium and europium and, to alesser extent, samarium and dysprosium.

As macropolycyclic compounds suitable for the purposes of the invention,it is possible to use the known cryptands, for example:

(1) The benzo-cryptands of the general formula: ##STR5## in which ○A ,○B and ○C independently of one another represent the groups 2_(B), 2 and1, which have the following meanings: ##STR6## in which X₁,2, Y₁,2 and Zeach represent a heteroatom chosen from the group consisting of oxygen,sulfur and nitrogen, it being possible for X₁ and X₂, and Y₁ and Y₂, tobe identical or different.

Examples of such cryptands which may be mentioned are those in which theunits ○A , ○B and ○C are respectively composed as follows:

○A ○B ○C =(2_(B) 11); (2_(B) 21); (2_(B) 22); (2_(B) 2_(B) 2); and thecompound of the formula: ##STR7##

(2) The cryptands containing nitrogen heterocycles, such as thosedescribed in JACS 99, 4583 (1977), of the formula I in which:

○A and ○B , which are identical, represent the polyethoxylated chain ofthe formula --(CH₂)₂ --O--(CH₂)₂ --O--(CH₂)₂ --, and

○C is a hydrocarbon chain containing a nitrogen heterocyclic as theenergy donor unit. Examples of such compounds which may be mentioned inparticular are the compounds below: ##STR8##

(3) The cryptands containing several nitrogen heterocycles as donorunits, such as the compound of the formula below: ##STR9## described inJ. Am. Chem. Soc. 1979, 101, p. 1047.

(4) The polycyclic cryptands containing aromatic units, for example thecompounds corresponding to the formula below: ##STR10## or the compoundsof the formula I in which the hydrocarbon chain carrying the donor unitor units is interrupted by a heteromacrocycle, such as the compounds ofthe formulae below: ##STR11##

Such compounds are described especially in: Angew: Chemie 86, 443(1974); Tet. letters 21, 941 (1980) and Chem. Comm. 833, 1981.

Other cryptands which can be used are the macropolycyclic compounds ofthe formula I above in which the molecular unit or units possessing atriplet energy greater than the energy of the rare earth to be cryptatedare chosen from the group consisting of phenanthroline, anthracene,bipyridines and the bisquinolines, possibly substituted is appropriatepositions and by groups capable of increasing the efficiency of thetransfer of energy or to modify the excitation spectrum of the rareearth cryptate. [J. Phys. Chem. 1964, vol. 68, p. 3324]. One example ofsuch groups is the phenyl group.

In these new macropolycyclic compounds, at least one of the radicals ○A, ○B or ○C preferably corresponds to one of the formulae below:##STR12##

A particular class of these macropolycyclic compounds consists of thecompounds of the formula I in which z is nitrogen, ○A and ○B are twomono- or polyethoxylated chains, preferably diethoxylated, and ○Ccorresponds to one of the formulae above.

These new macropolycyclic compounds also include the compounds of theformula I in which ○A and ○B each represent the group: ##STR13## and ○Cis one of the following groups: ##STR14##

Another particular class of these new macropolycyclic compounds isconstituted by the compounds of the formula I in which Z is nitrogen and○A , ○B , and ○C are identical and represent one of the above indicatedheterocycles, namely the 2,2'-bipyridine and phenanthroline.

The macropolycyclic compounds of the formula I in which the molecularmoiety or moieties are phenanthroline, anthracene, bipyridines orquinolines are new compounds with the exception of the compounds offormula I in which ○A and ○B are each a chain of the formula --(CH₂)₂--O--(CH₂)₂ --O--(CH₂)₂ -- and ○C is the group of the formula ##STR15##or the group of formula ##STR16## which are described in Chem. Ber. 111,pages 200-204 (1978).

The macropolycyclic compounds of the formula I can be obtained by knownchemical processes involving mainly condensation and/or additionreactions.

The processes described in French Pat. No. 70 21079 (2052947) and U.S.Pat. Nos. 3,888,877, 3,966,766 and 4,156,683 may be mentioned inparticular as examples of such processes. These processes are especiallysuitable for the preparation of compounds of the formula I in which Z isnitrogen or phosphorus.

To obtain compounds of the formula I in which Z is carbon and R is asdefined above, analogous condensation processes will be used.

In all cases, it suffices to use, as starting materials, chemicalcompounds each containing one of the radicals ○A , ○B or ○C and endsgroups capable of being su or containing radicals which can easily beremoved.

By way of example, it may be indicated that the compound of the formulaI in which Z is carbon, R is the hydroxyl group and ○A , ○B and ○C eachrepresent 2,2'-bipyridine can be obtained by condensing6,6'-dilithiobipyridine with 6,6'-dicyano-2,2'-bipyridine, hydrolyzingthe cyano group of the macrocycle thus obtained and condensing theresulting product with 6,6'-dilithiobipyridine.

In a preferred procedure, the new macropolycyclic compounds of theformula I in which Z is nitrogen and ○A and ○B are polyethoxylatedchains can be obtained by reacting:

the nitrogen macrocycle consisting of the two polyethoxylated chains,with

the hydrocarbon chain containing the said molecular unit, the said chainhaving cleavable end groups such as, for example, halogeno groups.

This coupling reaction is advantageously carried out in an anhydroussolvent, for example dimethyl sulfoxide (DMSO) or acetonitrile, ifappropriate in the presence of a reducing agent, such as sodium hydrideor sodium carbonate. The macropolycyclic compound is then obtained inthe form of a sodium salt.

The reaction is preferably carried out at a temperature below theboiling point of the solvent, for example at between 60° and 100° C.

It is possible, in order to obtain the free macropolycyclic compound toreact said sodium salt with silver nitrate to form the macropolycyclicsilver complex, which is thereafter treated with a stream of H₂ S. Theformed precipitate is then neutralized with a solution of (N(CH₃)₄ OHand extraced with methylene chloride.

It will be noted that the macropolycyclic rare earth complex accordingto the invention may be obtained from the free complex or from a saltthereof, such as the sodium salt for example.

In the above process, any one of the nitrogen-containing monocyclicmacrocycles described in U.S. Pat. No. 3,966,766 can advantageously beused as the macrocycle, the preferred compounds being:1,7,10,16-tetraoxa-4,13-diazacyclooctadecane of the general formula:##STR17## or 1,7,16-trioxa-4,13-diazacyclodecane of the general formula:##STR18##

It is pointed out that the preferred complexes according to theinvention, obtained from the above macrocycles, can be considered asderivatives of cryptates of the type 222 or 221, in which the etherunits in at least one of the hydrocarbon chains have been substituted byenergy donor units (in general aromatics or polyaromatics possiblycontaining heteroatoms).

The europium cryptates of type 222 or 221 have very low rates ofdissociation in aqueous solutions, although their formation constant isrelatively low (K=10⁶.8 and 10⁵.9 or the cryptates 221 and 222respectively--Inorganic Chem. 1981, 20, p. 616 and J.A.C.S. 1980, 102,p. 2278. Such cryptates do not satisfy the stability criteria defined inthe prior art. On the other hand, substitution of the ether units byenergy donor units, which provides the preferred complexes according tothe invention, does not modify the dissociation characteristic of thesecryptates but makes it possible, by excitation of the energy donor unitin its absorption band, to obtain a greatly enhanced fluorescencecharacteristic of the cryptated rare earth.

If the rare earth cryptates forming the subject of the present inventionare used specifically to label biologically active molecules with theaid of a covalent bond, they can be substituted on one or more of theirconstituent atoms by one or more sufficiently accessible substituentspossessing one or more molecular units which permit covalent couplingwith the biological molecule under operating conditions compatible withits biological integrity.

Non-limiting examples of these molecular units which may be mentionedare alkylamino, arylamino, isothiocyano, cyano, isocyano, thiocyano,carboxyl, hydroxyl, mercapto, phenol, imidazole, aldehyde, epoxide,thionyl halide, sulfonyl halide, nitrobenzoyl halide, carbonyl halide,triazo, succinimido, anhydride, halogenoacetate, hydrazino,dihalogenotriazinyl and other radicals (Biol. Chem. 245, 3059 (1970).The length of the arm bonding the macrocyclic complex to the molecule ofbiological interest can vary from 1 to 20 atoms, for example, and cancontain carbon atoms as well as heteroatoms such as N, O, S and P. Theinvention therefore also relates to the biological complexes consistingof a biological molecule which is associated by coupling or adsorptionwith a macropolycyclic complex according to the invention.

The coupling can be carried out using any of the reagents described forthis purpose in the literature and the labeled molecule can be separatedfrom the unreacted macropolycyclic complex by any suitable means ofseparation (for example gel filtration).

Among the molecules of biological interest which can advantageously belabeled with the rare earth cryptates forming the subject of the presentinvention, there may be mentioned, without implying a limitation,antigens, antibodies, monoclonal antibodies, fragments andantibody-fragment combinations, drugs, receptors, hormones, hormonereceptors, bacteria, steroids, amino acids, peptides, vitamins, viruses,nucleotides or polynucleotides in hybridization methods, enzymes andtheir substrates, lectins, nucleic acids, DNA and RNA.

The macropolycyclic rare earth complexes according to the presentinvention find an important application as fluorescent tracers inimmunological determinations, either in the so-called methods ofdetermination by competition or in the so-called methods ofdetermination by excess, in the homogeneous or heterogeneous phase, thesaid determinations being described in the prior art (LANDON, Ann. Clin.Biochem. 1981, 18, p. 253 and SOINI, Clin. Chem. 25, 353, 1979).

In the heterogeneous methods, it is possible advantageously to use:

tubes coated with antibodies specific for the substance to bedetermined, and to read off the fluorescence by the methods describedabove, directly through the tube Clin. Chem. 29, 60, 1983).

or a different solid phase, in particular a narrow strip or a gelatinousfilm on which a medium containing a specific antibody has been depositedbeforehand, the fluorescence being read off at a different angle fromthe excitation and the reflection of the exciting wave or directlythrough the support, if this is transparent.

Because of the line spectrum of these tracers, it is also possible todetect several antigens simultaneously either by using cryptates ofdifferent rare earths whose fluorescence lines do not overlap (forexample Tb and Eu), or by using conventional fluorescent tracers(fluorescein or rhodamine) and tracers according to the presentinvention.

Another application relates to immunochemistry, where the fluorescenceof the labeled cell is detected by microscopy in the manner described byR. C. NAIRN in "Fluorescent Protein Tracing, Longman Group Ltd.", 1976,which also has the possibility of carrying out multi-detection.

Another application of the rare earth complexes according to theinvention relates to cytology and cell classifiers, where the use of atracer having a line spectrum at high wavelength, coupled with the useof conventional tracers, makes it possible to perform multi-parameteranalyses.

Furthermore, with a given cryptand and different rare earth ions, it ispossible, with a single exciting wavelength, which is that of themolecular unit transferring the energy and which can be generated with asingle source (for example a laser), to obtain two fluorescence linespectra characteristic of the two cryptated ions (for example Tb and Eu)and thus disclosing the respective biological molecules to which thecryptates are fixed.

Another application relates to the use of the rare earth cryptatesaccording to the present invention in the field of genetic engineering,for example as indicators in hybridization reactions such as thosedescribed in European Patent Applications Nos. A-0 070 685 and A-0 070687.

The invention will now be described in greater detail by means of thenon-limiting illustrative examples below.

EXAMPLE 1 A--Preparation of (22)phenanthroline (hereafter called:(22)phen) ##STR19##

The solvent used in this synthesis is DMSO, which was dried for severaldays over a 4 Å molecular sieve and, if appropriate, distilled in vacuo.

1 mmol (366 mg) of dry and freshly chromatographeddibromomethylphenanthroline compound di-CH₂ Br-phen) was dissolved in 10to 20 ml of DMSO. It was transferred to a dry dropping funnel; thevolume was made up to 100 ml with dry DMSO.

100 to 200 mg of NaH in oil (FLUKA) were placed in a dry round-bottomedflask; 5 ml of dry toluene (or hexane) were added to the mixture, whichwas left to settle. The solution was evaporated in vacuo (1 torr) for 1hour. The NaH was redissolved in 20 ml of dry DMSO (evolution of H₂),with heating to 50° C. if appropriate.

1 mmol of the N₂ O₄ macrocycle (262 mg) was dried and 10 to 20 ml of dryDMSO were added.

The solutions of NaH and N₂ O₄ macrocycle were each introduced into adropping funnel and the volume was made up to 100 ml with dry DMSO.

100 ml of dry DMSO were placed in a three-necked round-bottomed flask.This flask was equipped with the two dropping funnels, a refluxcondenser and a drying system (silica gel). The flask was heated to60°-100° C., with magnetic stirring.

The contents of the two dropping funnels were simultaneously addeddropwise over a period of 1 to 2 hours.

The reaction mixture was heated for 1 hour after the reactants had beenadded. The mixture was then distilled in vacuo to remove the solvent.

It is possible to add a small quantity of water in order to remove thehydrides and the bases.

The reaction mixture was reduced to dryness to give a dry or pasty redresidue.

100 to 200 ml of CH₂ Cl₂ were added; the paste was triturated at roomtemperature for at least 30 minutes. The residue was filtered off andwashed. The organic liquid phase was retained and evaporated in vacuo.The residue obtained was dried in vacuo (1 torr, at least 30 minutes).

100 ml of ethyl ether were added to the residue, the components weremixed and the mixture was left to settle; this operation was repeated 3to 4 times.

To remove the remainder of the monocyclic N₂ O₄ macrocycle morecompletely, 50 ml of hexane and 1 ml of CH₃ OH were added to theresidue: the solution was evaporated in vacuo (gentle heating untilturbidity appeared; the solution was decanted and the operation wasrepeated several times.

The red residue was tested by slab chromatography (Polygram Alox N/UV₂₅₄from Macherey-Nagel); migration in the medium CH₂ Cl₂ /CH₃ OH 9/l gavethe following values:

    ______________________________________                                        (22)phen               Rf = 0.4-0.8                                           Monocyclic N.sub.2 O.sub.4 macrocycle                                                                Rf = 0.2-0.5                                           ______________________________________                                    

The residue was chromatographed on a column (20 cm, φ 15 cm) packed withneutral activated aluminum oxide 90 of 70-230 mesh (Merck), the eluentconsisting of the following mixtures: CH₂ Cl₂ /CH₃ OH (5%), CHCl₃ /C₂ H₅OH (5%), CH₃ OH/hexane 9/l. Yields from 12 to 35% according to theoperating conditions.

This gave the sodium salt of (22)phen, of the formula [Na⁺C(22)phen-Br.H₂ O], the elemental analysis of which was as follows:

    ______________________________________                                        found:    C 53.00       H 6.21  N 9.44                                        calculated:                                                                             C 53.16       H 6.18  N 9.55                                        ______________________________________                                    

B--Formation of the complex [Eu³⁺ C(22)phen]

0.07 mol (19.4 mg) of anhydrous EuCl₃ was dissolved in 4 ml of dry CH₃CN and the solution was heated under reflux for 1 hour 30 minutes. 35 mgof the previously obtained sodium salt of (22)phen in 2.5 ml of CH₃ CNwere added. The mixture was heated under reflux for 2 hours 30 minutes.After the mixture had been left to stand overnight at 4° C., the yellowprecipitate was filtered off.

The precipitate collected (11 mg) exhibits a strong fluorescence andthis also applies when it is in solution in H₂ O and CH₃ OH (UV 254 nm).

No change in the excitation and emission spectra was observed afterthree months in aqueous solution at a concentration below 10⁻⁴mol/liter.

EXAMPLE 2 A--Preparation of (22)phenanthrolinamide (hereafter called(22)phenamide) ##STR20##

The solvent used in this synthesis is acetonitrile distilled over P₂ O₅.The process was carried out in a dry glass apparatus.

305 mg of dry recrystallized di-COCl-phen (1 mmol) were dissolved in 50ml of CH₃ CN: after one night, the solution was filtered and transferredto a dropping funnel; the volume was made up to 90 ml with CH₃ CN.

524 mg of (22) (2 mmol) were dried in (<1 torr; overnight). The productwas dissolved in 90 ml cf CH₃ CN and transferred to a dropping funnel

The two dropping funnels were fitted to a 2 to 3 liter round-bottomedflask containing 1 liter of CH₃ CN. The two reactants were addedsimultaneously over a period of 2 hours to 2 hours 30 minutes, withrapid stirring (magnetic). Stirring was continued for 30 minutes afterthe addition. The solvent was removed in vacuo at 50° C. The residue wasdried for at least 1 hour at P<1 torr. The residue was mixed for 30minutes in 300 ml of CH₂ Cl₂ and filtered off. The filtrate wasevaporated to dryness. The residue was chromatographed on neutralactivated Alox 90 (column 30 cm, φ 1.5 cm), the eluent being CH₂ Cl₂containing 1% of CH₃ OH. The first product eluted was recovered.

310 mg of (22)phenamide were recovered with a yield of 63%. The productwas recrystallized from a mixture of toluene/hexane 1/l.

Melting point: 300°-302° C.

IR amide band at 1630 cm⁻¹

Mass spectrum: M⁺ at 494 for MW=494.55

    ______________________________________                                        Elemental analysis: C.sub.26 H.sub.30 N.sub.4 O.sub.6                                      C           H      N                                             ______________________________________                                        Calculated:        63.15       6.11 11.33                                                         59.8        5.7  10.5                                     Found:                                                                                           59.9        5.9  10.6                                      ______________________________________                                    

B--Formation of the complex [Eu³⁺ C(22)phenamide]

45 mg (0.17 mmol of anhydrous EuCl₃ were dissolved in 10 ml of dry CH₃CN. A solution of 80 mg (0.16 mmol) of (22)phenamide in 2 ml of CH₂ Cl₂was heated under reflux for 3 hours, 50 ml of CH₃ CN were added and themixture was heated to the boiling point of the CH₃ CN (CH₂ Cl₂ isremoved by evaporation).

Then the solution of Eu³⁺ was added.

The mixture was heated under reflux for 2 hours and then left to standat room temperature. The white precipitate was recovered by filtration(50 mg). It exhibited a strong red fluorescence (λ excitation: 254 nm)and the same applied in solution in H₂ O, CH₃ OH and DMSO.

    ______________________________________                                        Elemental analysis for EuC(22)phenamide(OH)Cl.sub.2.3H.sub.2 O                C.sub.26 H.sub.37 N.sub.4 O.sub.10 Cl.sub.2 Eu MW = 788.47                                 C           H      N                                             ______________________________________                                        Calculated:        39.61       4.73 7.11                                                          40.1        4.9  7.2                                      Found:                                                                                           40.3        4.8  7.1                                       ______________________________________                                    

No changes in the excitation and emission spectra of this complex wereobserved after three months in aqueous solution at a concentration below10⁻⁴ mol/liter.

EXAMPLE 3 Preparation of (22)anthracenamide ##STR21##

In this example, the method of high dilution is used.

0.564 g of the acid dichloride l (1.7 mmol) was dissolved in 70 ml ofanhydrous CH₂ Cl₂ and the solution was introduced into a 100 ml droppingfunnel;

0.446 g of the macrocycle (22), i.e. 1.7 mmol, and 0.47 ml oftriethylamine (2×1.7 mmol) were also dissolved in 70 ml of anhydrous CH₂Cl₂ and the solution was introduced into a 100 ml dropping funnel.

0.4 ml of toluene and 0.15 liter of anhydrous methylene chloride wereintroduced into a 3 liter three-necked flask. The two reactants preparedabove in the dropping funnels were introduced simultaneously into theflask over a period of 5 hours (14 ml/hour).

The organic phase was collected, concentrated to a few milliliters andthen transferred to a column of SiO₂ under pressure (column diameter 3.5cm; height 17 cm: eluent CH₂ Cl₂ /1% of methanol).

This gave a pale yellow, crystalline fluorescent product.

Yield: 45%

Thin layer chromatography (TLC): solvent CH₂ Cl₂ /2% of methanol; SiO₂ ;Rf=0.6

    ______________________________________                                        .sup.1 H NMR: solvent CDCl.sub.3 /CD.sub.2 Cl.sub.2 5/5, at 200               ______________________________________                                        MHz                                                                           ppm     2.53 (m)                                                                      2.93 (m)                                                                      3.18 (m)        24 H, HCH.sub.2 + OCH.sub.2 crown                             3.42 (m)                                                                      4.02 (d)                                                                      δ.sub.A = 4.62                                                                          J.sub.AB = 16 Hz 4H OCCH.sub.2 N                              δ.sub.B = 5.03                                                  7.6 (m)         4H                                                            8.43 (d)        2H             aromatic                                       8.82 (d)        2H                                                            ______________________________________                                        Microanalysis: C.sub.30 H.sub.36 O.sub.6 N.sub.2                              ______________________________________                                        Calculated:                                                                             C 69.20       H 6.97  N 5.38                                        Found:    C 69.07       H 7.00  N 5.22                                        ______________________________________                                    

Molecular weight: 520.55

The acid dichloride l used as the starting material in this example canbe obtained by the processes described by M. W. Miller et al. in R. W.Amidon and P. O. Tawney, J.A.C.S. 77, 2845 (1955) or by B. M. Mikhailov,Izvest. Akad. Nank. SS. Osdel Khim. Nank. 1948, 420-6; CA 42, 6350.

EXAMPLE 4 Preparation of (22)anthracene ##STR22##

380 mg of the diamide 3 (0.73 mmol) were partially dissolved in 20 ml ofannydrous THF contained in a round-bottomed flask.

The flask, fitted with a reflux condenser, was placed under an argonatmosphere and 8 ml of 1.1M B₂ H₆ in anhydrous THF were added at roomtemperature.

The whole reaction mixture was kept under reflux overnight and theexcess diborane was then destroyed at 0° C. with a few drops ofdistilled water.

The solvents were evaporated off and 40 ml of a 6N solution of HCl wereadded to the residual white solid.

The solution was heated under reflux for 30 hours under an argonatmosphere and became dark green.

It was left to cool, the water was then evaporated off and the resultingsolid was pumped dry with a vane pump for 1 hour and was then dissolvedin 50 ml of distilled water; 50 ml of CH₂ Cl₂ were added, the two phaseswere shaken and the aqueous phase was then separated off and renderedbasic at 0° C. with an aqueous solution of LiOH to pH 13; a solidprecipitated. A further 50 ml of CH₂ Cl₂ were added and the two phaseswere shaken and left to settle. The organic phase was recovered anddried over MgSO₄.

The crude product was transferred to a column of alumina and eluted withCH₂ Cl₂ /1% of methanol. A pure crystalline product was recovered on aTLC Plate.

Yield: 62-70%

TLC: Al₂ O₃ ; eluent CH₂ Cl₂ /6% of MeOH; Rf: 0.33

Melting point: >260° C.

    ______________________________________                                        .sup.13 C NMR: solvent CDCl.sub.3                                             ppm     25.2 (aromatic CH.sub.2)                                               54.3                                                                                          NCH.sub.2 macrocycle + branch                                56.3                                                                           69.3                                                                                          OCH.sub.2 macrocycle                                         70.0                                                                           ##STR23##                                                                    .sup.1 H NMR: solvent CDCl.sub.3                                              ppm     2.39 (t) 8H NCH.sub.2 macrocycle                                               ##STR24##                                                                     ##STR25##                                                                    3.03 and 3.82 (t) - 2 × 4H CH.sub.2CH.sub.2                      7.47 and 8.31 (AB) - 4H                                                                                 aromatic H                                         7.50 and 8.36 (AB) -  4H                                                      Microanalysis: C.sub.30 H.sub.40 O.sub.4 N.sub.2 (molecular weight            492.6)                                                                        Calculated:                                                                              C 73.13      H 8.18  N 5.6                                         Found:     C 73.06      H 8.04  N 5.2                                         ______________________________________                                    

The complex [Eu³⁺ C(22)anthracene] was prepared by following theproduced described in Example 1.

EXAMPLE 5 Preparation of the macropolycyclic compound of the formula (7)and the corresponding europium complex ##STR26## A--Preparation of6,6'-bis-bromomethyl-2,2'-bipyridine ##STR27##

A mixture of 6,6'-dimethyl-2,2'-bipyridine (2.76 g, 15 mmol) andN-bromosuccinimide (5.10 g, 28.6 mmol) in CCl₄ (150 ml) is heated underreflux for 30 minutes. Benzoyl peroxide (30 mg) was subsequently addedto the mixture, this was heated under reflux again for 2 hours and thesuccinimide was then filtered off. The solution was cooled to 0° C. andthe solid was filtered off and washed with methanol to give thebisdibromide in the form of a white crystalline solid (1.65).

Yield 32%; melting point 180°-181° C.

The CCl₄ solution was concentrated and chromatographed on a column(silica gel) by elution with a mixture of methylene chloride/methanol98/1 to give:

6,6'-bis-dibromomethyl-2,2'-bipyridine 0.9 g, 12%

6,6'-bis-bromomethyl-2,2'-bipyridine 1.38 g, 27%

6-methyl-6'-bromomethyl-2,2'-bipyridine 0.55 g, 14%

B--Preparation of the macropolycyclic compound

A mixture of the bis-hipyridine macrocycle of the formula (5) (0.15 g,0.38 mmol) (described in J. Org. Chem. 1983, 48, 4848) and Na₂ CO₃ (0.4g) in acetonitrile (200 ml) was heated to the reflux temperature and asolution of 6,6'-bis-bromomethyl-2,2'-bipyridine (0.12 g, 0.38 mmol) wasthen added over a period of 3 hours. The mixture was subsequently heatedunder reflux for 20 hours. The Na₂ CO₃ was filtered off and the filtratewas evaporated. The residue was filtered on a short column of alumina byelution with CH₂ Cl₂ /MeOH 98/2 to give the sodium salt of thetris-bipyridine macrobicyclic compound in the form of a white solid(0.16 g : 73%); melting point above 270° C.).

    ______________________________________                                        Microanalysis: C.sub.36 H.sub.30 N.sub.8, Na Br (677,6)                       ______________________________________                                        calculated:                                                                             C 63.81       H 4.46  N 16.53                                       found:    C 63.79       H 4.48  N 16.49                                       ______________________________________                                        NMR .sup.1 H: solvent CDCl.sub.3                                              ______________________________________                                        3.85 (s, 6CH.sub.2);                                                          7.33 (dd, J = 7,2; 1,2; 6H; H--C(5); H--C(5'))                                7.82 (t, J = 7.2; 6H; H--C(4); H--C(4'))                                      7.90 (dd; J = 7.2; 1.2; 6H; H--C(3); H--C(3'))                                ______________________________________                                    

A mixture of silver nitrate AgNO₃ (15 mg, 0.08 mmol ) and of the sodiumsalt obtained above (20 mg, 0.03 mmol ) was heated with 5 ml of CH₃ OHfor 30 minutes. The methanol was evaporated and the resulting complexwas purified over a column of silica gel with CH₂ Cl₂ /CH₃ OH as eluent(96/4).

The resulting silver complex (20 mg) was dissolved in a water-methanolmixture (1:1-5 ml) and treated with a stream of H₂ S for 15 minutes. Theformed precipitate was centrifuged and the solution neutralized withN(CH₃)₄ OH 0.1N and extracted with methylene chloride (3.5 ml). Thesolution was dried on MgSO₄ and evaporated; the resulting solid wasfiltered on silica gel CH₂ Cl₂ : CH₃ OH (96:4) to give the free complex(13 mg; 86%) with the following characteristics:

    ______________________________________                                        NMR .sup.1 H: solvent CDCl.sub.3                                              ______________________________________                                        3.82           (s, 12H, CH.sub.2)                                             7.44                                                                          7.78           ABX System J = 8; 7.5 and 0.9 Hz                               7.83                                                                          Microanalysis: C.sub.36 H.sub.30 N.sub.8 (574.7)                              ______________________________________                                        Found:       75.37         H 4.98                                                                              N 19.41                                      calculated:  C 75.24       H 5.26                                                                              N 19.50                                      ______________________________________                                    

The macropolycyclic compound obtained was used to prepare the complex[Eu³⁺ C(tris-bipyridine macropolycycle)] by following the proceduredescribed in Example 1.

EXAMPLE 6 Preparation of the bis-bipyridinephenanthrolinemacropolycyclic compound of the formula (9) and the correspondingeuropium complex ##STR28##

0.158 mmol of the bis-pyridine macrocycle (5) was weighed into a 500 mltwo-necked round-bottomed flask. 0.75 mmol of Na₂ CO₃ (approx 5-foldexcess) and 105 ml of freshly distilled CH₃ CN were added. The mixturewas heated under reflux for 30 minutes, with magnetic stirring.

An equimolecular dibromophenanthroline solution in 100 ml ofacetonitrile was then added dropwise.

Refluxing and magnetic stirring wet continued throughout the rather slowaddition (2 hours 10 minutes).

The mixture was then heated for a further 18 hours under the sameconditions. The solution obtained was evaporated to dryness. The crudeproduct, dissolved in CH₂ Cl₂, was washed with water. (Thin layerchromatography on alumina:eluent CH₂ Cl₂ /MeOH 90/10.)

This crude product was purified on a column of standardized alumina(activity II-III), the eluent being CH₂ Cl₂ /MeOH 98/2.

    ______________________________________                                        Microanalysis: C.sub.38 H.sub.30 N.sub.8, Na Br, H.sub.2 O                    ______________________________________                                        (719,6)                                                                       calculated:                                                                              C 63.42     H 4.45  N 15.57                                        found:     C 62.77     H 4.46  N 15.31                                        ______________________________________                                        RMN .sup.1 H: solvent - CDCl.sub.3                                            ______________________________________                                        3.91 (s, 8H, CH.sub.2 -bpy)                                                   4.07 (s, 4H, CH.sub.2 -phen)                                                  7.36 (dd, J = 7.2; 1,3; 4H; H--C(5); H--C(5') of                              the bipyridine)                                                               7.64 (d, J = 8.2; 2H; H--C(3), H--c(8) of phenanthroline,                     7.78 (s, 2H, H--C(5), H--C(6) of phenanthroline                               7.83 (t, J = 7.2; 4H; H--C(4); H--C(4') of bipyridine                         7.91 (dd, J = 7.2; 1,3; 4H; H--C(3); H--C(3') of bipyridine                   8.27 (d, J = 8.2; 24; H--C(4);                                                4-C(7) of phenanthroline.                                                     ______________________________________                                    

The macropolycyclic compound thus obtained was used to prepare thecomplex [Eu³⁺ C(bis-biyrinephenanthroline macropolycycle)] by followingthe procedure described in Example 1.

The excitation and emission wavelengths characteristic of this complexare shown in the Table below.

EXAMPLE 7

The macropolycyclic complexes below were obtained by following theprocedure of Example 1 using the macropolycyclic compounds(22)pyridinamide and (222_(B)) respectively:

    [Eu.sup.3+ C(22)pyridinamide]

    Tb.sup.3+ C(222.sub.B)]

EXAMPLE 8 Preparation of the (22)bisisoquinoline macropolycycliccompound of the formula (12) and of the corresponding europium complex.##STR29## (a) preparation of 1,1-bis(bromomethyl)-3,3' biisoquinoline ofthe formula 10

This compound were prepared from 1-methyl-3-hydroxyisoquinoline offormula ##STR30## The 1-methyl-3-hydroxyisoquinoline was prepared bycyclization of N-(.sup.±)-phenyl ethyl-diethoxyacetamide. 72 g of thisstarting compound, prepared according to the method of preparationdescribed by H. FUKUMI et al in "HETEROCYCLES" 9(9) 1197 (1978) andpurified by distillation under reduced pressure (140° C. 0.1 mmHg), werepoured dropwise under stirring for one hour into 400 ml of concentratedH₂ SO₄ cooled down to 10° C. The reaction mixture was cooled during theaddition, so that its temperature does not exceed 10° C. The reactionmixture was then stirred for ten hours at room temperature, then pouredinto 600 g of ice. After filtration, the clear solution was neutralizedwith 20% aqueous medium under efficient cooling. The yellow precipitatewas filtered off, washed with water and dried in vacuo. 41.5 g of1-methyl-3-hydroxyisoquinoline were obtained (yield 90%). Said compoundwas then tosylated. A suspension of 41.5 g of1-methyl-3-hydroxyisoquinoline was then stirred with pyridine (250 ml)under cooling in an ice-water bath. P-toluenesulfonyl chloride (70 g,1.5 equivalent) was then gradual added over a period of 30 minutes. Theyellow starting compound has then disappeared. The reaction wasmonitored by TLC. As soon as the reaction was complete, water (50 ml)was added, and the stirring was continued for 1 hour. It was thendiluted with 700 ml of water and neutralized with solid Na₂ CO₃. Theresulting precipitate was filtered off, carefully washed with water anddried. 70.5 g of a non-hydroscopic product, stable in air was obtained(yield : 86%).

The resulting 1-methyl-3-p-toluenesulfonyloxyisoquinoline was thencoupled according to the method of preparation described by M. Tiecco etal in SYNTHESIS 736, 1984.

In a three-necked flask equipped with a magnetic stirrer, flushed withargon, 1 of dimethylformamide (DMF), 236.3 g of triphenylphosphine and53.5 g of NiCl₂, 6H₂ O were introduced, forming a deep blue-greesolution. When the temperature of the oil bath reached 50° C., 14.64 gof zinc powder were added and the colour of the solution changed to abrown-red colour. After one hour, the solution of the starting product(70 5 g in 200 ml of DMF), namely the1-methyl-3-p-toluenesulfonyloxyisoquinoline, was quickly added dropwisewith stirring, under heating at 50° C., said temperature was maintainedfor additional 6 hours. After cooling down to room temperature, thereaction mixture was poured into 4 of diluted ammonia (1.51 of H₂ O and500 ml of 20% NH₃). A vigourous air stream was bubbled through thesuspension to oxidize the Ni(Ph₃ P)₄ (Ph=phenyl) The end of oxydationwas indicated by the disappearance of the brown colour The suspensionwas filtered off, washed with water and placed in 400 ml of 20% HCl tochange the biisoquinoline into its water-insoluble hydrochloride. Thesuspension was then shaked with two portions of 400 ml of ethyl ether toremove the triphenylphosphine and the acid layer was filtered off,washed with 100 ml of water and acetone to remove traces of Ph₃ P andPh₃ PO. The hydrochloride was placed in a round-bottomed flask with 200ml of 20% ammonia and stirred overnight to release the base. The whiteproduct thus obtained (26 g), namely the1,1-dimethyl-3,3'-Biisoquinoline was filtered off, carefully washed withwater and dried in vacuo (yield 81%) This product was poorly soluble inmost of the solvents and sparingly soluble in CH Cl₃ and THF The1,1-dimethyl-3,3'-biisoquinoline thus obtained was then brominated toform the 1,1'-bis (bromomethyl)-3,3'-biisoquinoline.

1.20 g of 1,1'-dimethyl-3,3'-biisoquinoline was dissolved in 500 ml ofCCl₄ under reflux and N-bromosuccinimide (2.26 g, 3 equivalents) wasadded. After ten minutes, 10 g of 2,2'-azobis (2-methylpropionitrile)were added. The reaction was monitored by thin layer chromatography. Twoother parts of initiator (10 mg each) were added over a period of onehour. After three hours, the solution was evaporated to dryness and theresidue was treated with 150 ml of methanol, stirred for 30 minutes andfiltered off. The resulting solid was washed with 100 ml of methanol.The filtration cake was dried in vacuo, dissolved in boiling toluene(100 ml) and quickly filtered. The product precipitated during the nightin the refrigerator (1.28 g-yield:68%).

(b) Preparation of the macropolycyclic compound of the formula 12

To a mixture under stirring of 1.416 g of N₂ O₄ macrocycle, and 3.39 gof Na₂ CO₃ in 150 ml of CH₃ CN, a suspension of 1,1'-bis(bromomethyl)-3,3'-biisoquinoline in CH₃ CN (100 ml) was added over aperiod of 3 hours. Stirring was continued for 20 hours. After filtrationand washing with CH₃ CN and evaporation, a crude product was obtainedand chromatographed twice, first over alumina (eluents : 3% CH₃ OH inCHCl₃ then 10% CH₃ OH in CHCl₃ V/V) then over silica gel (eluent 10% ofCH₃ OH in CHCl₃). The yield after two purifications was of 0.289 g,namely 14%. Another purification was carried out by cristallization in amixture ethanol-ether by vapour diffusion.

(c) Preparation of the complex [Eu³⁺ C(22)biisoquinoline]

24.5 mg (1 equivalent) of anhydrous europium nitrate were dissolved in0.5 ml of anhydrous CH₃ CN. The sodium complex of (22) biisoquinoline(37.0 mg) in 0.3 ml of CH3CN was added and the resulting mixture wastreated by following the method disclosed in example 2b. The pale yellowprecipitate thus obtained was diluted in 3 ml of acetonitrile and heatedfor 2 hours. The solution was allowed to stand at room temperature forseveral days. A yellow crystallized product of the formula 12 wasobtained.

EXAMPLE 9: Preparation of the (22) diphenylbipryidine macropolycycliccompound of the formula 18 ##STR31## (a) Preparation of 6,6'-bis(bromomethyl)-4,4'-diphenyl-2,2'-bipridine (16)

This compound were prepared from the 4,4-diphenyl 2,2'-bipryidineavailable on the market, according to the following method.

6,6'-dimethyl-4,4'-diphenyl-2,2'-bipryidine

To a suspension under stirring of 4,4'-diphenyl-2,2'-bipyridine (4 g, 13mmol) in anhydrous tetrahydrofuran (THF), cooled with an ice-bath, wasadded dropwise 1,5M methyllithium (3 equivalents) under nitrogenatmosphere. After this addition, the solution was stirred for additional30 minutes under the same conditions. The dark red mixture thus obtainedwas then heated and stirred at 40° C. for 3 hours under nitrogen.

An excess of water was slowly added at 0° C. under nitrogen. The organicphase was separated and the aqueous phase was extracted three times withdichloromethane . Manganese dioxide was then added (20 to 40 times theweight of the starting compound) to the orange organic solution. Themixture was stirred at room temperature for 30 to 50 minutes. Thereaction was followed by thin layer chromatography (Al₂ O₃ -eluent:toluene). Magnesium sulfate was then added to the reactionmedium, which was stirred for 30 additional minutes. The filtrate wasthen filtered off and evaporated. The residue was chromatographed overalumina (eluent:toluene/hexane 1:1) and two products were obtained: the6,6'-dimethyl-4,4'-diphenyl-2,2'-bipyridine (0.85 g;19%); the6-monomethyl-4,4'-diphenyl-2,2'-bipyridine(0.45 g:10.8%)

6,6'-dimethyl-4,4'-diphenyl-2,2'-bipyridine-N,N'-dioxide

In a 250 ml flask containing an ice-cooled stirred solution of the abovecompound (0.28g, 0.83 mmol) in chloroform (60 ml), it was graduallyadded a solution of m-chloroperbenzoic acid (0.57 g 3.3 mmol) inchloroform (60 ml). Stirring was continued for 3 hours, during which themixture was left to return to room temperature. The reaction mixture wastreated with an aqueous solution of sodium bicarbonate (3.3 mmol)stirred for 30 minutes.

The organic phase was separated and evaporated in vacuo. The residue wasre-dissolved in dichloromethane (CH₂ Cl₂) and passed over a column ofbasic alumina. A second chromatography was carried out on standardalumina to completely purify the product (0.21 g; 68%).

6,6'-bis(acetoxymethyl)-4,4-diphenyl-2,2'-bipyridine

0.21 g (0.57 mmol) of the above compound was heated for one hour underreflux in acetic anhydride (1.3 ml). The solution was concentrated invacuo and toluene was added until the formation of azeotrope of themixture.

The resulting solid was re-dissolved in dichloromethane, washed with a10% aqueous solution of sodium bicarbonate, dried and the solvent wasevaporated. The crude product was passed over a column of silica gel andeluted with dichloromethane to give 0.126 g (yield : 49%) of the desiredcompound

6,6'-bis(bromomethyl)-4,4'-diphenyl-2,2'-bipyridine(16)

A solution under stirring of the above compound (0.053 g; 0.117 mmol)and of 47% hydrobromic acid (0.9 ml) was heated for 4 hours at 130° C.

The solution was then cooled in a methanol/ice-bath . 15 ml of water, 40ml of chloroform, then gradually a saturated solution of sodiumcarbonate, were added thereto, until the pH of the solution becamealkaline.

The organic phase was separated and the aqueous phase was extracted withchloroform (2×10 ml). All the organic fractions were combined andevaporated. The residue was chromatographed over an alumina column, withthe chloroform as eluent. 40 mg (yield:69%) of the dibromide of theformula 16 were obtained.

(b) Preparation of salt [Na⁺ C(22) diphenyl-bipyridine Br] of theformula 18

The method described in example 5b was followed by using:

21.2 mg (0.081 mmol) of N₂ O₄ macrocycle of the formula 17

43 mg (0.40 mmol) of sodium carbonate

40 mg (0.081 mmol) of dibromide of the formula 16

29.8 mg (yield:53%) of the complex of the formula 18 were obtained.

EXAMPLE 10 Preparation of the diphenylbipyridine-bisbipyridinemacropolycyclic complex of the formula 19 ##STR32##

The method of the above example was repeated by using the bis-pyridinemacrocycle of the formula (5) (example 5) instead of the N₂ O₄macrocycle of the formula 17. There were used:

24.7 mg (0.063 mmol) of 5 bis-pyridine macrocycle

31.1 mg (0.063 mmol) of dibromide of the formula 16

0.1 mg (0.94 mmol) of sodium carbonate

The reaction lasted for 23 hours and the compound of the formula 19 wasobtained with a yield of 20%.

EXAMPLE 11 Preparation of the biisoquinoline bis-bipyridinemacropolycyclic compound of the formula 20 ##STR33##

The method of the example 8 was repeated by using the bis-bipyridinemacrocycle of the formula 5 instead of the N₂ O₄ macrocycle of theformula 11. Equimolecular quantities of the compound of the formula 5and of dibromide of the formula 10, were used, and the compound of theformula 20 was obtained (31.7 g ; yield : 20%)

EXAMPLE 12 Preparation of the (22) bipyridine macropolycyclic compound

Following the method of example 5 and using the N₂ O₄ macrocycle of theformula 11 instead of bisbipyridine of the formula 5 and themacropolycyclic compound of the formula 21 was obtained with a yield of12%. ##STR34##

EXAMPLE 12

Also using the method of operation of Example 1 with the macropolycycliccompounds obtained according to examples 9 to 11, the followingmacrocyclic complexes were respectively obtained:

    [Eu.sup.3+  C(22) bipyridine]

    [Eu.sup.3+  C (bpy.biisoq.)]

    [Eu.sup.3+  C (22) diph.bpy]

    [Tb.sup.3+  (22) bipyridine]

Likewise, by operating according to the method of example 5, the complex[Eu³⁺ C(macropolycycle tris-phenanthroline)] was prepared by using the6,6'-bis-bromomethyl-phenanthroline and the diamine bis(phenanthrolinediyl) macrocycle. The corresponding macropolycycliccompound had the following characteristics:

    ______________________________________                                        microanalysis: C.sub.42 H.sub.38 N.sub.8, NaBr, H.sub.2 O                     ______________________________________                                        (767.6)                                                                       calculated:                                                                             C 65.71       H 4.17  N 14.6                                        found:    C 65.23       H 4.26  N 13.1                                        ______________________________________                                        RMN 1.sub.H : solvent CDCl.sub.3                                              ______________________________________                                        4.03 4.45 (very broad; AB, 12H, CH.sub.2)                                     7.66 (d, J = 8.1; 6H; H--C(3); H--C(8))                                       7.78 (s, 6H, H--C(5); H--C(6))                                                8.27 (d, J = 8.1, 6H; H--C(4); H--C(7))                                       ______________________________________                                    

fluorescent characteristics of the cryptates of the invention (a)excitation peaks for a given emission wavelength

For each complex was determined, for a given emission wavelength,characteristic of the rare earth ion of the complex, the excitationwavelengths the recorded excitation peaks do not correspond to those ofthe rare earth ion alone In addition, it has been noted that by excitingthe complex at the wavelength corresponding to the excitation peakdetermined above, the maximum of fluorescence was characteristic of therare earth ion.

The above determinations were made with spectrometer PERKIN-ELMER LS 5in the operating conditions indicated in table I hereunder.

(b) Measurement of the life-time τ of europium and terbium cryptates.

On a spectrometer LS5, the emission spectrum of the peak situatedbetween 610 and 620 nm for europium, and between 540 and 550 forterbium, was recorded, the solution being excited in one of theadsorption peaks. It was operated in phosphorescence method, the valueof the slit being fixed to 1 ms=tg.

Recordings were made for several values of td (time limit) namely 0.1 ;0.2; 0.3 ; 0.4 and 0.5ms.

The intensity of the peak It was measured and τ was determined by theformula: ##EQU2## by tracing the curve log I_(t).

I_(o) may be determined and τ may be calculated as a function of td. Theobtained results are indicated in table II hereunder.

                                      TABLE I                                     __________________________________________________________________________    EXCITATION AND EMISSION CHARACTERISTICS OF THE COMPLEXES OF THE               INVENTION                                                                     (Perkin-Elmer LS 5)                                                                                                           EMISSION                                          Width of    EXCITATION           fluorescence                         Concentration                                                                         the   Expansion                                                                           given λ                                                                     recorded λ                                                                        given λ                                                                     maximum                  Complex     (solvent)                                                                             slits factor                                                                              emission                                                                           excitation excitation                                                                         observed                 __________________________________________________________________________                                                         at:                      [Eu.sup.3+ C(22)phen]                                                                     3.7 · 10.sup.-6 mol/l                                                        2.5/20 nm                                                                           10    615 nm 290 nm(peak)                                                                           290 nm  615 nm(peak)                      (water)                    312 nm                                                                        (shoulder)      593 nm                                                        330 nm          (second                                                       (shoulder)      peak)                  [Eu.sup.3+ C(22)phenamide]                                                                2.5 · 10.sup.-4 mol/l                                                        2.5/10 nm                                                                           3     613 nm 290 nm(peak)                                                                           290 nm 613 nm                             (water)                    323 nm(peak)                                                                  333 nm                                                                        (shoulder)      590 nm                                                        345 nm                                                                        (shoulder)                             [Eu.sup.3+ C(tris-bi-                                                                     7 · 10.sup.-7 mol/l                                                          2.5/10 nm                                                                           15    617 nm maximum at                                                                             300 nm maximum at             pyridine macro-                                                                           (0.1 M phos-               300 nm          617 nm                 cycle)]     phate buffer,                                                                 pH 7.5)                                                           [Eu.sup.3+ C(phenanthroline-                                                              2.8 · 10.sup.-5 mol/l                                                        2.5/10 nm                                                                           2                     280 nm 590 nm                 bis-bipyridine)]                                                                          (water)                                    613 nm                  [Eu.sup.3+ C(22)bipyri-                                                                   10.sup.-5 M                237 nm(peak)    617 nm                                    2.5/10 nm                                                                           10    617 nm          310 nm                        dine]       (water)                    303 nm(peak)    592 nm                 [Eu.sup.3+ C(22)biiso-                                                                    10.sup.-5 M                                                                           2.5/10 nm                                                                           0.5   617    355 nm(peak)                                                                           325 nm 617 nm                 quinoline   Tris buffer                325 nm(peak)                                       0,01 M pH                                                         [Eu.sup.3+ C(22)biphe-                                                                    10.sup.-5 M                                                                           2.5/10 nm                                                                           5     617 nm 254 (peak)                                                                             320 nm 617 nm                 nylbipyridine]                                                                            H.sub.2 O                  323 (peak)      592 nm                 [Eu.sup.3+ C(bis-bipy-                                                                    0.06 mg/                                                                              2.5/10 nm                                                                           20    617 nm 310 (peak)                                                                             310 nm 617 nm                 ridine biiso-                                                                             2.4 ml of                                  592 nm                 quinoline)] water                                                             [Eu.sup.3+ C(22) anthra-                                                                  0.2 mg in                                                                             2.5/10 nm                                                                           0.2   613 nm 287 nm(peak)                                                                           287 nm 590 nm(peak)           cene]       3 ml of water              317 nm(shoul-   613 nm(peak)                                                  der)                                   [Eu.sup.3+ C(22)bipyridine                                                                2 · 10.sup.-4 mol/l                                                          2.5/5 nm                                                                            40    --     --       290 nm 613 nm                 amide]*     (DMSO)                                     590 nm                 [Tb.sup.3+ C 222.sub.B ]**                                                                10.sup.-3 mol/l                                                                       2.5/20 nm                                                                           15    541 nm 290 nm(shoulder)                                                                       290 nm 541 nm                             (DMSO)                     333 nm(peak)    485 nm                 [Eu.sup.3+ C(tris-phenan-                                                                 0.3 mg/3 ml                                                                           2.5/10 nm                                                                           5     617 nm 272 (peak)                                                                             270 nm 617 nm                 throline)]  of Tris buffer                             593 nm                 [Tb.sup.3+ C(22)bipyri-                                                                   0.1 mg/2.4 ml                                                                         2.5/10 nm                                                                           1     617 nm 240 (peak)                                                                             320 nm 545 nm                 dine]       H.sub.2 O                  305 (peak)      492 nm                                                        314 (shoulder)  586                    __________________________________________________________________________                                                           nm                      *Measurement made in the phosphorescence mode; delay td = 0.1 millisecond     tg = 2 milliseconds                                                           **Measurement made in the phosphorescence mode; delay td = 0.2 millisecon     tg = 5 milliseconds                                                      

                                      TABLE II                                    __________________________________________________________________________                    λexcitation                                            Macropolycyclic complex                                                                       nm    medium                                                                              concentration                                                                        τms                                    __________________________________________________________________________    [Eu.sup.3+ C(22)phen]                                                                         280   H.sub.2 O                                                                           3.7 · 10.sup.-4 M                                                           0.27                                       [Eu.sup.+3 C(22) phen amide]                                                                  280   H.sub.2 O                                                                           2.5 · 10.sup.-6 M                                                           0.21                                       [Eu.sup.3+ C(22)bipyridine]                                                                   310   H.sub.2 O                                                                           1.2 · 10.sup.-5 M                                                           0.41                                       [E.sup.+3 C(22)biisoquinoline]                                                                325   tris  10.sup.-5 M                                                                          0.135                                                            0 01 M                                                                        pH 7                                                    [Eu.sup.3+ C(22)biphenylbipyridine]                                                           320   H.sub.2 O                                                                           10.sup.-5 M                                                                          0.32                                       [Eu.sup.3+ (tris-bipyridine)]                                                                 300   Phosphate                                                                           2 · 10.sup.-7 M                                                             0.43                                                             buffer                                                                        0.01 M                                                                        pH 7.4                                                  [Eu.sup.3+ (bis-bipyridine-                                                                   311   H.sub.2 O                                                                           10.sup.-5 M                                                                          0.25                                       biisoquinoline]                                                               [Eu.sup.3+ C(tris-phenanthroline)]                                                            272   tris  10.sup.-4 M                                                                          0.32                                                             0.01 M                                                                        pH 7                                                    [Tb.sup.3+ C(22)bipyridine]                                                                   318   tris  4 · 10.sup.-5 M                                                             0.72                                                             0.01 M                                                                        pH 7                                                    __________________________________________________________________________

We claim:
 1. A macropolycyclic compound corresponding to the formula:##STR35## in which Z is a trivalent or tetravalent atom; R is nothing orrepresents hydrogen, the hydroxyl group, an amino group or a hydrocarbonradical; and the divalent radicals A, B and C independently of oneanother are selected from the group consisting of hydrocarbon chains,which hydrocarbon chains with one or more heteroatoms and interrupted byheteromacrocycle, wherein at least one of the radicals A, B and Cincludes or consists essentially of a radical selected from the groupconsisting of ##STR36## provided that when Z is N and A is radical 5 or6, then B and C may not simultaneously be --(CH₂)₂ --O--(CH₂)₂--O--(CH₂)₂ --.
 2. A compound of claim 1 in which at least one of theradicals A, B, and C includes or consists essentially of radical 1, 2,or
 5. 3. A compound of claim 2 wherein Z is nitrogen and A and B arepolyethoxylated chains.
 4. A compound of claim 1 in which at least oneof the radicals A, B, and C includes or consists essentially of radical3, 4, or
 6. 5. A compound of claim 4 wherein Z is nitrogen and A and Bare polyethoxylated chains.
 6. A compound of claim 1 wherein Z isnitrogen and A and B are polyethoxylated chains.
 7. A compound of claim1 which is a tris-bipryidine macropolycycle of the formula ##STR37## ora phenantrholine-bis-bispryidine macropolycycle of the formula ##STR38##8. A compound of claim 1 which is a tris-phenanthroline macropolycycle.9. A compound of claim 1 which is a bis-bipyridine bi-isoquinolinemacropolycyle of the formula ##STR39## or a bis-bipyridinediphenylbipyridine macropolycycle of the formula ##STR40##
 10. Amacropolycyclic compound of the formula ##STR41## in which A is selectedfrom the group consisting of ##STR42##